Activation of Dopamine D2 Receptor Alleviates Neuroinammation and Neuronal Injury in Mice Model of Allergic Rhinitis With Olfactory Dysfunction

Background: Allergic rhinitis (AR) is a common chronic allergic disease of the upper airway that not only causes peripheral inammation, but also induces neuroinammation in the hippocampus, prefrontal cortex, olfactory bulb and other brain areas. Recent studies have suggested that the dopamine D2 receptor acts as a key target in regulating immune functions and neuroinammatory reaction, which may be a promising target for AR-induced olfactory dysfunction (OD). Methods: An AR mouse model with OD induced by ovalbumin (OVA) were constructed. A coculture system of olfactory bulb neurons (OBNs) and microglias was established. The buried food pellet test was to evaluate the olfactory function of the mice. Immunouorescence staining, HE staining, ELISA, Western blotting, and TUNEL staining were also used to investigate the molecular mechanisms underlying the anti-inammatory effects of the dopamine D2 receptor in AR-induced OD. Results: We conrmed that AR mice with or without OD had the characteristics of AR, but the expression of the microglial marker CD11b and the related cytokines (TNF-α, IL-1β and IL-6) in the AR mice with OD were signicantly increased in the olfactory bulb compared with those of mice without OD. Nasal administration of quinpirole, a dopamine D2 receptor agonist, shortened the time to nd the food pellets, inhibited the expression of TLR4/MyD88/NF-κB signalings and the levels of TNF-α, IL-1β and IL-6. In the coculture system of OBNs and microglias, quinpirole inhibited the release of TLR4/MyD88/NF-κB signalings-dependent inammatory cytokines in the microglias, which was accompanied by decreased AMPA receptor GluR1, increased GluR2 and reduced TUNEL positive cells in the OBNs. Conclusion: Activation of the dopamine D2 receptor inhibits the release of inammatory cytokines through the microglia-dependent TLR4/MyD88/NF-κB signalings, alleviates AMPA receptor-mediated damage of the olfactory bulb, and protects olfactory function. on neuroinammation in the olfactory bulb and olfactory function in vivo. Additionally, we established a coculture system of olfactory bulb neurons (OBNs) and microglias, and investigated the mechanism of quinpirole reversal of OBN damage in vitro. Our results clearly demonstrated that activation of dopamine D2 receptor improves microglia-dependent neuroinammation and restores olfactory damage. microglia-related depolarization of nucleus Our results found that the of the dopamine D2 inhibited the release of and accompanied with increased and decreased thereby alleviating the excitotoxicity mediated by AMPARs in the olfactory bulb. modulates microglial


Introduction
Allergic rhinitis (AR) is a common clinical otorhinolaryngology disease that has a huge impact on public health. Olfactory dysfunction (OD) is a common concomitant symptom of AR, which impairs the ability to discriminate avors, detect poisonous and pernicious gas from the environment [1]. Olfactory bulb, as a relay station for olfactory transmission, plays a crucial role in olfactory formation. Actually, microgliosis are representative pathological changes that develop during neuroin ammation of central nervous system (CNS) [2]. A close link between microglial hyperplasia of olfactory bulb and olfactory dysfunction has been reported in the patients and animal models of the neuroimmunological disorders, including experimental autoimmune encephalomyelitis (EAE), Alzheimer's disease (AD) and Parkinson's disease (PD) and so on [3][4][5]. Furthermore, recent studies have shown that airway allergen exposure directly induces neuroin ammation [6,7], such as increased Th2-type cytokines and nNOS in the olfactory bulb of airway in ammation animal models [8,9]. Therefore, alleviating neuroin ammation (like microglial hyperplasia) of the olfactory bulb may be expected to improve AR-induced OD.
As an important catecholamine neurotransmitter, dopamine can be used as bridge molecule to connect the nervous and immune systems [10]. Recently, the anti-neuroin ammatory effects of the dopamine D2 receptor have been highlighted, and its agonists prevent the degeneration of dopaminergic neurons by inhibiting the TLR4/NF-κB pathway in PD mice [11]. Furthermore, the microglia-dependent TLR4/NF-κB pathway is a key link in inducing neuronal AMPA receptor (AMPAR) tra cking and subsequent excitotoxicity injury [11], which plays an important role in brain damage in many in ammatory diseases, such as peripheral in ammatory pain, autoimmune encephalopathy, brain injury, in ammatory bowel disease and others [12][13][14][15]. For example, the activation of glial purinergic signals by TLR4 can signi cantly increase the excitatory synaptic drive of CA1 neurons by modulating glutamate receptor tra cking [16]. Activation of TLR4 induces an increase in microglia-dependent TNF-α release, which can recruit calcium-permeable AMPAR into synapses [17]. In addition, excitability of the cerebral dentate gyrus after brain injury accompanied by increased TLR4 signaling is mediated by the synaptic AMPAR current, while the NMDAR, another glutamate receptor, does not change [18]. Consistent with this, blocking of TLR4 signaling after brain injury can reduce the calcium-permeable AMPAR current of granular cells in the dentate gyrus, reduce neural network excitability and epilepsy susceptibility [19,20]. Based on the above studies, we speculate that AR-induced OD may be accompanied with AMPAR-mediated neuronal damage induced by microglia activation, and dopamine D2 receptor agonists are expected to reverse this process.
In this study, we used an ovalbumin (OVA)-induced AR model with OD to detect microglial in ammation related indicators, and explored the effect of the dopamine D2 receptor agonist quinpirole on neuroin ammation in the olfactory bulb and olfactory function in vivo. Additionally, we established a coculture system of olfactory bulb neurons (OBNs) and microglias, and investigated the mechanism of quinpirole reversal of OBN damage in vitro. Our results clearly demonstrated that activation of dopamine D2 receptor improves microglia-dependent neuroin ammation and restores olfactory damage.

Materials And Methods
Establishment of the AR model with olfactory dysfunction The 6-8 week old mice were provided by the Animal Experiment Center of Renmin Hospital of Wuhan University. All experimental procedures were approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University (License No. WDRM 20190419). The AR model with OD was established as described previously [21]. On days 0, 7, and 14, 300 µL PBS containing 50 µg OVA (grade V; Sigma) and 1 mg of aluminum hydroxide was administered by intraperitoneal injection for sensitization. On the 21st day, 20 µL PBS containing 400 µg OVA was administeredere intranasally once a day for 2 consecutive weeks for the challenge. The mice in the control (Ctr) group were given the same amount of PBS in the nasal cavity. Finally, the AR mice with OD were selected by the buried food pellet test (Fig. 1a).

Buried food pellet test
The buried food pellet test (BFPT) was used to evaluate the olfactory function of mice as previously described [22]. Brie y, the mice were fasted 18-24 hours before the test. In the test cage (42 cm length × 28 cm width × 18.5 cm height), a food pellet of approximately 4 g was buried at a depth of 0.5 cm under the bedding. After 1 hour of habituation, the time it took the mice to grasp the food pellets with their front paws or teeth was recorded. If the food pellets were not found within 300 seconds (an average of three tests), it was identi ed as olfactory dysfunction: the AR mice with OD were selected based on this criterion. In addition, we measured the latency time of each mouse after the drug treatments.

Drug treatment and experimental grouping
The AR mice with OD were identi ed and randomly divided into the following groups: quinpirole (Quin, 3 mg/kg, Sigma) was administered nasally every day for 3, 6, 9, and 12 days, and the control group was given PBS in equal doses. The drug concentration and route of administration used was based on the previously published article [23], and the nasal-brain pathway allows the drugs to bypass the blood-brain barrier and enter the central nervous system. During this period, the mice was challenged with OVA every other day. The olfactory function was evaluated 24 hours after the administration, and the mice were used for the following experiments after being sacri ced (Fig. 3a).

Coculture of olfactory bulb neurons and microglias
The use of the Transwell co-cultivation system and magnetic bead sorting maintains the in vivo state of the microglias to a greater extent, and additionally recapitulates the relationship between microglias and neuroin ammation. As previously reported [24,25], the microglias were isolated from the olfactory bulbs after successful modeling. The olfactory bulbs were enzymatically digested, and ltered through 70 µm cell strainers to prepare the single cell suspension. The microglias were sorted with CD11b magnetic beads (Miltenyi Biotech). After 3 rounds of resuspending, loading, and washing, the positive cells on the sorting column were collected. Furthermore, the olfactory bulbs of mice were isolated within 3 days after birth, and digested with 0.25% pancreatin at 37 °C for 15 minutes after removing the meninges and blood vessels. The cells were resuspended with DMEM/F12 medium containing 10% FBS (Gibco), inoculated into a polylysine-coated culture plate, and cultured a humidi ed CO 2 (5%) incubator at 37 °C. After 7 days, they were co-cultivated with puri ed microglias for 24 hours, and were simultaneously treated with quinpirole (20 µmol/L).

HE staining
After the mice were sacri ced, their noses were xed in 4% paraformaldehyde for 48 hours, decalci ed in 20% EDTA for 2 weeks, and then made into para n sections. After the para n sections were depara nized, hematoxylin was used to stain the nucleus and eosin was used to stain the cytoplasm. The morphology of the mouse nasal mucosa was then observed under a light microscope (× 400); 5 elds on each slice were randomly selected, in which the number of eosinophils was counted under the microscope, and then averaged.

Immuno uorescence staining
The olfactory bulbs were xed with 4% paraformaldehyde, embedded in slices, and baked. After the para n sections were completely dewaxed with xylene, 10% calf serum was added, and the section were placed at room temperature for 10 minutes. The sections were incubated with rabbit polyclonal anti-CD11b antibody (1:500, Abcam) at 4 °C overnight, followed by a FITC-labeled goat anti-rabbit IgG (Abcam) at room temperature for 30 minutes. The sections were washed with water, blown dry, sealed with glycerin, and followed by observation under a uorescence microscope (Olympus). TUNEL staining TUNEL staining was carried out according to the manufacturer's instructions (Servicebio). The cells were xed with 4% paraformaldehyde for 30 minutes, ruptured by 0.2% Triton X-100 for 5 minutes, and incubated with 50 µl TUNEL reaction solution at 37 °C for 60 minutes in the dark. DAPI was used to stain the nucleus. The cells were observed and photographed under a uorescence microscope (Olympus). In 6 non-repeating high-power (× 400) elds, the number of TUNEL-positive cells as a percentage of the number of DAPI-positive cells was used to calculate the positivity rate.

ELISA
The olfactory bulbs, culture supernatant and serum were collected after drug treatments, and the levels of in ammatory mediators were measured using ELISA kits of TNF-a, IL-6, IL-1β and OVA-speci c IgE (BD Biosciences) according to the manufacturer's instructions, respectively. The experiments were repeated for three times.

Western blot
After the drug treatment, the total protein was extracted. RIPA lysis buffer containing a protease inhibitor and a phosphatase inhibitor was added to the tissues or cells, which were then placed on ice for 30 minutes. After centrifugation at 12000 g for 15 minutes at 4 °C, the supernatant was extracted. The surface proteins were extracted according to the instructions of the Cell Fractionation Kit (Thermo Fisher). The protein concentration was measured by a BCA kit (Absin), and samples of 40 µg were loaded onto the gel for SDS-PAGE electrophoresis. After SDS-PAGE electrophoresis, the proteins were transferred to a membrane, which was blocked in 5% skim milk for 1.5 hours. The following primary antibodies, which were incubated at 4 °C in a shaker overnight, were used: TLR4 (1:500, Santa Cruz), MyD88 (1:500, Santa Cruz), NF-κB P65 (1:1000, Santa Cruz), NF-κB p-P65 (1:1000, Santa Cruz), GluR1 (1:1000, Abcam), GluR2 (1:2000, Abcam), and β3-tubulin (1:1000, Abcam). The secondary antibody was labeled with HRP (1:50000, Servicebio) and incubated with the membrane at room temperature for 1 hour. The blots were washed 3 times for 10 minutes. The bands were visualized with enhanced chemiluminescence (ECL, Millipore) using a gel imaging system (Bio-Rad). ImageJ was used to determine the gray value. Using β3tubulin as the reference protein, the relative intensities of each target protein band were calculated.

Statistical analysis
All results were represented as mean ± SEM. The data and graphs were analyzed by GraphPad Prism 8.0 (GraphPad Software, USA). The results were analyzed by one-way ANOVA followed by post hoc Tukey's tests for multiple comparisons. A p value < 0.05 was considered signi cant.

Results
Establishment of the mouse model of AR with olfactory dysfunction To verify this model, histopathology, OVA-speci c IgE and allergic symptoms were evaluated, and the AR mice were divided into groups with or without OD by the buried food pellet test (BFPT). The incidence of OD observed was 68.46%. HE staining showed that the in ltration of eosinophils was signi cantly increased in the submucosa of the AR mice with and without OD (Fig. 1b, c). The OVA-speci c IgE levels of the AR mice with and without OD were signi cantly increased (Fig. 1d), and the frequency of sneezing and rubbing of these mice was also signi cantly increased (Fig. 1e). This nding suggests that the AR mice with or without OD exhibit the characteristics of AR, and the AR mice with OD selected met the needs of the subsequent experiments.
Hyperactivity of the microglia from the olfactory bulb in AR mice with olfactory dysfunction To verify the relationship between AR-induced OD and neuroin ammation in the olfactory bulb, we detected the expression of microglial marker CD11b and the related cytokines TNF-α, IL-1β and IL-6 in the olfactory bulb. Compared with the group without OD, the expression of CD11b, TNF-α, IL-1β and IL-6 in the group with OD increased by 94.78% (P < 0.01), 596.81% (P < 0.01), 281.65% (P < 0.01) and 247.78% (P < 0.01), respectively (Fig. 2a, b, c). Similarly, the expression of TLR4, MyD88, and NF-κB p-P65/P65 in the OD group increased by 86.10% (P < 0.01), 45.67% (P < 0.05) and 99.08% (P < 0.01), compared with group without OD, respectively (Fig. 2d, e). However, the expression of the above proteins in the group without OD group was not signi cantly different from that of the control (Ctr) group. These results suggest that olfactory dysfunction is involved in microglia hyperactivity of the olfactory bulb and is related to the TLR4/NF-κB pathway.

Disccussion
Several studies on humans and rodents have shown that the intranasal or nebulized inhalation in allergen-sensitized animals induced avoidance behavior and activated limbic brain areas [8,9,32,33]. OVA or pollen-induced AR rats produced TH2 cytokines in the olfactory bulb and prefrontal cortex but not in the temporal cortex and hypothalamus, and increased brain activity was observed by functional MRI [8]. Asthma induces activation of the microglias in the hippocampus and prefrontal cortex, elevated levels of TNF-α and IL-1β, and a signi cant loss of neurons in the brain [33]. In the brain responses observed in allergic asthma, atopic dermatitis and multiple sclerosis, the levels of CCL11 increase, which promote eosinophil in ltration and subsequent neuronal damage in the affected area, followed by facilitation of the migration of microglias and ROS production, which ultimately enhance neurotoxicity induced by glutamate [34][35][36]. Additionally, the choroid plexus is an important structure of the ventricle with bloodbrain barrier (BBB) permeability, which has a high level of expression of IL-4Rα. Macrophages of the choroid plexus that respond to IL-4 can release pro-in ammatory cytokines, which then leak into the brain to promote the microglia to produce a second wave of cytokines [37]. Endothelin-1 produced by in ammatory tissue may increase BBB permeability and activate microglia expressing endothelin receptor B through the damaged BBB [38]. Other hypotheses cite the role of IL-1β in allergic reactions, which activates the hypothalamic-pituitary-adrenal (HPA) axis, stimulates the release of cortisol and serotonin, and leads to mood disorders [39]. This study also showed that AR-induced OD was closely related to neuroin ammation of the olfactory bulb. The expression of microglia marker protein CD11b, TNF-α, IL-1β and IL-6 in the olfactory bulbs were signi cantly increased in the AR mice with OD, which is similar to the ndings in the hippocampus [32]. However, the mechanism of allergen-induced damage to the olfactory bulb is not clear.
Numerous studies have shown that TLR4 is a key molecule that regulates the immune response during CNS infection and injury [26]. After activation in the brain, TLR4 binds to MyD88 to relieve the inhibitory effect of IκB on NF-κB, promote NF-κB nuclear translocation, stimulate in ammation-related gene expression, and promote the synthesis and release of TNF-α, IL-1β and IL-6 [26][27][28]. Our research found that the dopamine D2 receptor agonist quinpirole inhibited the expression of TLR4 and downstream signal molecules in the olfactory bulb, and the release of in ammatory cytokines. Similar to these results, quinpirole suppressed the expression of TLR4/NF-κB pathway in PD mice by increasing the expression of βArr2, thereby preventing dopaminergic neuron degeneration [11]. In addition, the regulatory effect of dopamine D2 receptor on neuroin ammation is also related to the mechanisms, such as NLRP3 in ammasome, renin-angiotensin system (RAS) and αB-crystallin. For example, the selective dopamine D2 receptor agonist LY171555 inhibited the activation of NLRP3 in ammasomes in the substantia nigra pars compacta of PD mice, thereby further controling the assembly process of the in ammasomes [40]. L-DOPA suppressed the production of angiotensinogen in astrocytes through the dopamine D2 receptor, thereby inhibiting microglia-mediated in ammation and neuronal oxidative stress caused by excessive activation of RAS [41]. Furthermore, quinpirole reduceed the level of pro-in ammatory mediators in the substantia nigra of PD mice by increasing the expression of αB-crystallin [42]. This study also suggests that dopamine D2 receptor activation can reduce the TLR4/NF-κB-dependent release of TNF-α, IL-1β and IL-6 in the microglia and alleviate the in ammatory response of the olfactory bulb.
Recent studies have shown that the microglia releases the pro-in ammatory cytokines TNF-α and IL-1β to participate in the regulation of AMPAR tra cking, which is a key link in inducing neuroin ammatory damage [29][30][31]. The AMPAR tra cking results in intracellular Ca 2+ overload, triggering a series of neurotoxic cascades such as mitochondrial damage, oxidative stress, and cell death [43,44]. In a cervical spinal cord contusion model, the number of GluR1-containing AMPARs at the ipsilateral synapse increased after injury. In vivo nanoinjection of TNF-α into the ventral horn of the spinal cord resulted in increased GluR1 and decreased GluR2 at extrasynaptic and synaptic plasma membrane sites [45]. In subsequent studies, the expression of the GluR1 subunit was increased in human NT2-N neurons exposed to TNF-α, leading to an increased susceptibility to kainate-induced necrosis, which was related to the A-Smase/NF-B pathway [46]. Similar ndings were observed in hippocampal neurons and lumbar motor neurons, in which TNF-α or IL-1β increased the surface expression of GluR1-containing AMPAR, accompanied by a signi cant increase in AMPAR-mediated excitatory postsynaptic currents [16,47]. IL-1β promote the release of NOS and presynaptic glutamate, and ultimately lead to enhanced AMPAR activity [48]. In addition, IL-1β in the brain is involved in microglia-related in ammatory pain and leads to the depolarization of paraventricular nucleus neurons or the membrane hyperpolarization of hypothalamic neurons, which is associated with abnormal AMPAR activation [49]. Our results found that the activation of the dopamine D2 receptor inhibited the release of TNF-α, IL-1β and IL-6, accompanied with increased GluR1 and decreased GluR2, thereby alleviating the excitotoxicity mediated by AMPARs in the olfactory bulb.
In summary, inhibiting neuroin ammation is a promising strategy in the treatment of neurological diseases. For example, quinpirole prevents neuroin ammation-mediated dopaminergic neuron degeneration in PD [11] and reduces microglia-mediated in ammation [41]. After the occurrence of intracerebral hemorrhage (ICH) or PD, exogenous quinpirole inhibit neuroin ammation and improve the outcome of the nervous system [23,42]. The dopamine D2 receptor agonist bromocriptine signi cantly inhibited the hyperactivity of glial cells and reduced the production of TNF-α in the spinal cord of amyotrophic laternal sclerosis (ALS), thereby preventing the loss of motor neurons [50,51]. Therefore, the dopamine D2 receptor may be an effective target to improve the neuroin ammation in the olfactory bulb, and ultimately help to develop drugs for the treatment of AR with olfactory dysfunction.

Conclusion
In conclusion, the microglias of olfactory bulb were abnormally activated in AR with olfactory dysfunction, followed by the upregulation of TLR4/MyD88/NF-κB signals, TNF-α, IL-1β and IL-6. Quinpirole, a dopamine D2 receptor agonist, can improve TLR4/MyD88/NF-κB signalings-dependent neuroin ammation and AMPAR-mediated exitotoxicity, thereby helps to restore olfactory function. Our ndings suggest an association among dopamine D2 receptor, neuroin ammation and AMPAR-mediated neuronal damage in olfactory bulb, providing a novel target for the treatment of olfactory dysfunction induced by AR.